Accurate diagnosis of tuberculosis infection is essential for the treatment, prevention and control of this resurgent disease. Since Mycobacterium tuberculosis (M. tb) is often difficult to identify in patients with active tuberculosis, and impossible to directly identify in healthy latently infected people, an immune-based diagnostic test indicating the presence or absence of M. tb infection is useful for diagnostic evaluation of active tuberculosis and diagnosis and screening of latent M. tb infection.
The first measure of the cellular immune response to be exploited as a marker of M. tb infection, developed at the end of the 19th century, was the tuberculin skin test (TST), which measures a delayed type hypersensitivity response to tuberculin purified protein derivative (PPD). This test has many drawbacks including poor specificity because of cross-reactivity of PPD, a crude mixture of over two hundred M. tb proteins widely shared between M. tb and M. bovis Bacille Calmette-Guerin (BCG) and most environmental mycobacteria. Hence false-positive results are common in people with environmental mycobacterial exposure and previous BCG vaccination. This presents a significant problem because most of the world's population is BCG-vaccinated and the confounding effect of BCG persists for up to 15 years after vaccination.
Comparative genomics has identified several genetic regions in M. tb and M. bovis that are deleted in M. bovis BCG. Several regions of difference, designated RD1 to RD16, between M. tb or M. bovis and BCG have been identified. All represent parts of the M. bovis genome deleted during prolonged in vitro culture. RD1 was deleted before 1921, when BCG was first disseminated internationally for use as a vaccine. RD1 is thus absent from all vaccine strains of BCG, as well as most environmental mycobacteria, but is still present in M. tb complex, including all clinical isolates of M. tb and M. bovis. There are nine open reading frames (ORFs) in the RD1 gene region. Early secretory antigen target-6 (ESAT-6) and culture filtrate protein 10 (CFP10) are encoded in RD1 and have been intensively investigated in animal models and humans over the last few years. ESAT-6 and CFP-10 are the strongest targets of the cellular immune response in M. tb-infected mice, cattle and tuberculosis patients and contacts. They are therefore now used as the key antigens in interferon-gamma release-assays (IGRAs) which exploit the fact that interferon-gamma-secreting T cells specific for ESAT-6 and CFP-10 are commonly detected in M. tb-infected persons but almost never detected in BCG-vaccinated persons.
The development and validation of such T cell-based interferon-gamma release assays (IGRAs) over the last decade is the first major advance in diagnosis of tuberculosis infection since the development of the tuberculin skin test 100-years ago. Although diagnostic sensitivity of commercially available IGRAs is higher than TST, their real-life clinical use demands higher sensitivity to enable rapid exclusion of active tuberculosis and reliably diagnose latent tuberculosis in those at highest risk of progression to tuberculosis and who are at risk of false-negative IGRA results, ie people who are immunosuppressed by virtue of HIV-infection, concomitant chronic illness (eg end-stage renal failure, diabetes, immune-mediated inflammatory diseases) medication (eg corticosteroids, anti-TNF-alpha agents) or young age (children under 5-years and especially under 2-years of age). One approach is to increase diagnostic sensitivity by incorporating additional antigens that are strong targets of T cell responses in M. tb-infected persons but not in BCG-vaccinated persons.
The Rv3615c gene is situated outside the RD1 locus and encodes a 103 amino-acid protein of unknown function. However, Rv3615c protein has been identified as a critical component of the secretion pathway called the Snm (secretion in mycobacteria) system (MacGurn et al Molecular Microbiology 2005 57:1653) involved in secreting the virulence factors ESAT-6 and CFP-10. This protein is not in the RD1 locus (which is absent in all strains of BCG vaccine) and thus is not expected to be specific for M. tuberculosis infection in BCG-vaccinated persons. This antigen is also not expected to be a strong target of T cell responses because the strongest T cell antigens in M. tb-infected humans are all secreted antigens and there is no available data to indicate that Rv3615c is secreted by M. tb. Moreover, there is no available evidence to indicate that Rv3615c is a T cell target in M. tb-infected humans.
Bovine TB, caused by Mycobacterium bovis (M. bovis), a pathogenic mycobacterium closely related to M. tb, is a major problem in UK cattle herds and results in a great economic burden. All cattle that are presumed to have M. bovis infection on the basis of positive skin test results in response to bovine tuberculin [PPD-B] are slaughtered because they cannot be used for milk or beef production and, if they develop active TB, become infectious to other cattle in the herd. Because of the great economic burden caused by bovine TB, veterinary researchers are actively investigating T cell immune responses in M. bovis-infected cattle in order to develop effective cattle vaccines and cattle diagnostics for improved prevention and early detection of bovine TB. With these objectives, a research group at the Veterinary Laboratories Agency in the UK investigated cellular immune responses to a range of M. bovis proteins that are highly expressed at the mRNA level during in vitro culture. As part of a screen of over 100 antigens, they found 14 genes that were strongly expressed at the mRNA level (Sidders et al. Infection and Immunity 2008 vol 76 (9); 3932-3939). On screening these proteins for T cell responses in cattle with presumed M. bovis infection, they found that 4 were T cell antigens, since they were recognised by IFN-gamma-secreting T cells from the cattle. The 4 M. bovis antigens were Mb2107c, Mb3299, Mb3776c and Mb3645c, and they gave IFN-gamma responses in 2, 3, 5 and 11 cows respectively out of 30 cattle (with presumed M. bovis infection) tested (Sidders et al. Infection and Immunity 2008 vol 76 (9); 3932-3939). The corresponding genes in M. tb are: Rv2081c, Rv3271c, Rv3750c and Rv3615c respectively. The screening used 20mer peptides representing the sequences of these 4 gene products in 30 cattle presumed to be naturally infected with M. bovis (on the basis that they had positive skin test results in response to bovine tuberculin [PPD-B]) from herds known to have bovine tuberculosis (Sidders et al. Infection and Immunity 2008 vol 76 (9); 3932-3939). Control cattle comprised 10 uninfected cattle obtained from herds in four yearly testing parishes with no history of bovine tuberculosis breakdown in the past 4 years (PPD-B skin test negative) and 20 cattle vaccinated with BCG Danish strain around 6 months prior to sampling. Although IFN-gamma responses to Mb3645c (Rv3615c) measured by whole blood ELISA were detected in 11/30 (37%) of the presumed M. bovis-infected cattle, no responses were detected to this antigen in either the naive (0/10) or BCG-vaccinated (0/20) cows. Responses to Mb3645c (Rv3615c) were identified in 4/7 M. bovis-infected cows that did not have interferon-gamma T cell responses to ESAT-6 and CFP-10. Thus, Mb3645c (Rv3615c) seems to be recognised by T cells from more cattle than the other 3 antigens, but this difference (11/30 vs 5/30) was not statistically significant (P=0.14, Fisher's exact test).
It is not possible to predict based on the antigen whether a T-cell antigen in cattle will also be a T-cell antigen in humans. There are a number of significant differences in antigen processing, presentation and recognition between cattle and humans. In addition, cattle have substantially different MHC molecules from humans, and are thus expected to recognise different antigens. Moreover, cattle are genetically more homogenous than out-bred human populations which are ethnically diverse and genetically heterogeneous. Accordingly, the skilled person would have no reason to consider a cattle antigen could be a T-cell antigen in other species.
As well as improving methods of diagnosis of M. tb, it would be useful to provide additional vaccines for M. tb. Although the immune mechanisms of protection against tuberculosis remain hitherto undefined, T cell-mediated immunity is essential for protection. All tuberculosis vaccine candidates currently in clinical trials (e.g. ESAT-6, MVA-85A, ESAT-6-Ag85B fusion molecule, recombinant BCG over-expressing 85A/Mtb10.4, Ad35 expressing 85A/85B/Mtb10.4) are based on M. tuberculosis antigens that elicit strong T-cell immunity during natural infection. A major challenge in vaccine development is to identify immunodominant antigens that elicit a strong IFN-gamma and IFN-gamma/IL-2 polyfunctional response from effector and memory T cells of both CD4 and CD8 T cell subsets. These vaccine antigens also need to be highly recognised in infected individuals across the human population to be immunogenic and effective in genetically heterogeneous out-bred populations.